MULTIPLE OPIATE BINDING-SITES IN THE CENTRAL NERVOUS-SYSTEM OF THE RABBIT - LARGE PREDOMINANCE OF A MU SUBTYPE IN THE CEREBELLUM AND CHARACTERIZATION OF A KAPPA SUBTYPE IN THE THALAMUS

  • 1 January 1983
    • journal article
    • research article
    • Vol. 24  (1) , 23-29
Abstract
The binding characteristics of [3H]etorphine, a nonselective .mu.-,.delta.- and .kappa.-opiate agonist, were compared with those of [3H]Tyr-D-Ala-Gly MePhe-NH(CH2)2OH ([3H]DAGO), a selective .mu.-agonist, in rabbit cerebellar and thalamic membranes. The ability of various unlabeled opioid ligands to compete with the binding of [3H]etorphine in the 2 preparations was examined in cerebellar membranes, [3H]DAGO(Kd = 0.7 nM) labels slightly fewer sites than does [3H]etorphine (Kd = 0.06 nM): 0.18 vs. 0.24 pmol/mg of protein. Competition studies indicate that up to 75% of the [3H]etorphine binding sites in this preparation display high apparent affinity for unlabeled DAGO and higher apparent affinity for morphine, the prototypical .mu.-agonist, than for Tyr-D-Ala-Gly-Phe-D-Leu (DADL), a .delta.-agonist. The rabbit cerebellum evidently contains a very high proportion (0.7-0.8) of .mu.-opiate binding sites. In thalamic membranes, [3H]DAGO (Kd = 1.1 nM) labels considerably fewer sites than does [3H]etorphine (Kd = 0.08 nM): 0.09 vs. 0.27 pmol/mg of protein. In this preparation, the competition curves of DAGO and of DADL resolve binding of [3H]etorphine into 2 components. The 1st component accounts for 40-50% of total binding and reflects the interaction of [3H]etorphine with .mu.-opiate binding sites. The 2nd component (up to 50% of total binding) is unaffected in the presence of DADL at concentrations (1-10 .mu.M) that rule out binding of [3H]etorphine to .mu.- and .delta.-opiate binding sites. It disappears readily in the presence of very low concentrations (Ki [inhibition constant] < 1 nM) of benzomorphan opiates (bremazocine, cyclazocine and ethylketocyclazocine) yet it is relatively insensitive to inhibition by .mu.- and .delta.-agonists. This 2nd component may therefore reflect [3H]etorphine''s interaction with a .kappa.-opiate binding site. The .kappa.-opiate binding site is assayed for as that site which binds [3H]etorphine (0.5 nM) in the presence of either DADL (2 .mu.M) or 10 .mu.M of another enkephalin: Tyr-D-Ser-Gly-Phe-Leu-Thr. In the rabbit CNS, the thalamus, followed by frontal cortex and caudate nucleus, shows the highest content of .kappa.-opiate binding sites.